Trypanosoma brucei, the African trypanosome, is a protozoan parasite that causes sleeping sickness in humans and a similar disease in livestock. This research aims to understand the molecular mechanisms involved in regulating replication of the mitochondrial DNA of African trypanosomes. Trypanosomes have long been known for a characteristic form of mitochondrial DNA termed kinetoplast DNA (kDNA). kDNA consists of a topologically interlocked network of several thousand minicircles and 20-30 maxicircles. The maxicircles encode mitochondrial ribosomal RNAs and genes for oxidative metabolism. In general, DNA replication is regulated at the level of initiation, not elongation. The recent discovery of T. brucei mitochondrial DNA primase gene PRI 1 and its expression as a recombinant protein will permit the development of an in vitro system for investigating the initiation of minicircle and maxicircle DNA replication. Prior studies of kDNA replication have relied on powerful, but indirect, methods such as RNA interference. Studies of kDNA replication using purified proteins has not been possible previously since in vitro synthesis by a DNA polymerase requires a primer, which is usually RNA. The availability of a mitochondrial DNA primase will form the basis of an in vitro system using recombinant proteins and plasmid templates containing minicircle or maxicircle replication origins. Many kDNA replication proteins have been identified based on immunolocalization and/or RNA interference of gene expression. In particular, the proteins UMSBP and P38 have been implicated in playing a direct role in initiation of DNA synthesis at the minicircle replication origin. Both proteins will be expressed as recombinant proteins and will be available as components of the initial minicircle replication system. This study will identify the enzymes and proteins required for initiating DNA synthesis of each of the two strands on minicircle and maxicircle origins and will identify precise sites of DNA strand initiation. Additional replication proteins will be identified based on their interaction with PRI 1 or PRI 2, a second putative mitochondrial DNA primase that will also be investigated. Both PRI 1 and PRI 2 have been found to be essential for both minicircle and maxicircle replication. Finally, the role of an essential mitochondrial DNA ligase in kDNA duplication will be determined based on overexpression of the ligase and analysis of resulting kDNA networks. Overall, this project will reveal molecular details of the mechanisms that regulate replication of the mitochondrial genome of this early diverging human parasite and will likely indicate possible new targets for drug development.